Methods to control protein structure and function inside living cells have proven to be valuable tools to elucidate the roles of proteins in their native biological contexts (Schreiber, 2003; Buskirk and Liu, 2005; Banaszynski and Wandless, 2006). Traditional genetic methods that have been widely used to control protein function by altering expression levels in mammalian cells include knock-out and knock-in systems such as those mediated by Cre-Lox recombination (Sauer et al., 1988) and the use of transcriptional regulators such as the tetracycline-responsive tet-on/tet-off systems (Gossen et al., 1992). These methods are highly specific to the protein of interest and can be applied to many proteins, but typically require days to reach steady-state protein levels in mammalian cells, are irreversible in the case of recombination-based methods, and are vulnerable to transcriptional compensation (Shogren-Knaak et al., 2001; Marschang et al., 2004; Wong and Roth, 2005; Acar et al., 2010). Other methods such as RNA interference (Fire et al., 1998), chemical genetics (Kino et al., 1987), small-molecule regulated protein stability or degradation (Stankunas et al., 2003; Schneekloth et al., 2004; Banaszynski et al., 2006), and small molecule induced proteolytic shunts (Pratt et al., 2007) have also been used effectively by many researchers and offer more rapid control over protein levels than strategies that exert control before transcription, but can require the discovery of small molecule modulators of protein function, necessitate the involvement of other cellular machinery that may not be present in the cells of interest, or are prone to off-target effects.
Protein-splicing elements, termed inteins, can mediate profound changes in the structure and function of proteins. Inteins are analogous to the introns found in polynucleotides. During intein-mediated protein splicing, inteins catalyze both their own excision from within a polypeptide chain and the ligation of the flanking external sequences (exteins), resulting in the formation of the mature protein from the exteins, and the free intein. No natural inteins, however, have been shown to be regulated by small molecules. Extein function is typically disrupted by the presence of an intein but restored after protein splicing. Many inteins can splice in foreign extein environments. Therefore, inteins are powerful starting points for the creation of artificial molecular switches.
Ligand-dependent inteins have been engineered (see, e.g., PCT application WO 2005/098043). Since inteins function in a variety of extein environment, ligand-dependent inteins are universally applicable to regulate the activity of a variety of target proteins in mammalian cells in a ligand-dependent manner without disturbing transcriptional or translational pathways. However, conventional ligand-dependent inteins were developed for use at room temperature and exhibit poor splicing efficiency or high background splicing in the absence of ligand when incubated at higher temperatures. These characteristics limit the application of ligand-dependent inteins in mammalian cells.